Cloning, expression and characterization of recombinant elastase from Pseudomonas aeruginosa in Picha pastoris.
The gene lasB from Pseudomonas aeruginosa, which encoded elastase, was cloned and firstly successfully expressed in Pichia pastoris stain KM71 under the control of AOX promoter. The effects on the recombinant elastase activities of different pH, different temperatures and different metal ions were assayed. The full-length gene (1497 bp) encodes a preproenzyme including an N-terminal signal peptide (23 aa), a propeptide (197 aa) and mature elastase (301 aa). The recombinant elastase was secreted into culture supernatants using signal sequence from lasB and showed a single band at about 34 kDa by SDS-PAGE. The recombinant elastase expression hit the highest level of approximately 450 mg/L and the specific elastolytic activity of the recombinant elastase was 130 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The optimal temperature and pH of the recombinant elastase was 28 degrees C and 7.4, respectively. The enzyme possessed high resistance to heat, and can be activated by Ca(2+). These enzyme properties suggested that it could be produced in an industrial scale and has the potential to be a commercial enzyme.
Lin
Xijin
X
Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Xu
Wentao
W
Huang
Kunlun
K
Mei
Xiaohong
X
Liang
Zhihong
Z
Li
Zhemin
Z
Guo
Jingxin
J
Luo
YunBo
Y
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa